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Purification and characterization of cytosolic pyruvate <span style="font-size:14.0pt;font-family:HiddenHorzOCR;mso-bidi-font-family: HiddenHorzOCR">kinase from developing seeds <i>of Brassica campestris L</i> </span>

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Title Purification and characterization of cytosolic pyruvate kinase from developing seeds of Brassica campestris L
 
Creator Singh, D K
Malhotra, Sarla P
Singh, Randhir
 
Description 51-58
Cytosolic pyruvate kinase (ATP: Pyruvate phosphotransferase,
EC 2.7.1.40; PKc) was purified to apparent homogeneity with about 22% recovery
from developing seeds of Brassica campestris using (NH4)SO4
fractionation,

DEAE-cellulose chromatography, gel filtration through
Sepharose-CL-6B and affinity chromatography through reactive Blue Sepharose-CL-6B.
The  purified enzyme with molecular mass
of about 214 kDa was a heterotetramer with subunit molecular mass of 55 and 57
kDa. The enzyme showed maximum activity at pH
6.8 and absolute requirement for a divalent

(Mg2+) and a monovalent (K+)
cation for activity. Typical Michaelis-Menten kinetics was obtained for both
the substrates with Km values of 0.10 and 0.11 mM for PEP and ADP, respectively. The enzyme could also
use UDP or GDP as alternative nucleotides, but with lower Vmax and
lesser affinities. The enzyme was inhibited by glutamate, glutamine, fumarate,
citrate,

isocitrate, oxalate, 2-PGA. ATP, UTP and GTP and
activated by glucose-6-phosphate, fructose-1,6-bisphosphate and Pi, suggesting
its regulation mainly by TCA cycle intermediates and the cellular need for
carbon skeletons for amino acid biosynthesis. ATP inhibition was of competitive
type with respect to PEP and non-competitive with respect to ADP.

Similarly, oxalate inhibition was also of competitive
type with respect to PEP and non-competitive with respect to ADP. Initial velocity
and product inhibition studies except for
pyruvate inhibition were consistent for a compulsory-ordered tri-bi mechanism.
 
Date 2012-12-28T11:32:50Z
2012-12-28T11:32:50Z
2000-02
 
Type Article
 
Identifier 0975-0959 (Online); 0301-1208 (Print)
http://hdl.handle.net/123456789/15371
 
Language en_US
 
Rights CC Attribution-Noncommercial-No Derivative Works 2.5 India
 
Publisher NISCAIR-CSIR, India
 
Source IJBB Vol.37(1) [February 2000]